The P-glycoprotein multidrug transport protein is a membrane glycoprotein which is overexpressed in cell lines and tumors which are resistant to structurally unrelated cytotoxic agents. The exact mechanism of action is unknown, however, the overexpression of the protein results in reduced uptake of cytotoxic drugs. We have been investigating the interaction of forskolin a naturally occurring diterpene, and derivatives of forskolin with the P-glycoprotein. Forskolin photoaffinity agents have been synthesized which are capable of covalent labeling the forskolin binding site. We have demonstrated that the binding site may be identical to that identified by other labeling compounds such an 3H-azidopine and azido derivatives of prazosin. However, the forskolin photoaffinity labels are much more efficient and specific at labeling the P-glycoprotein than other photoaffinity agents. The location of the forskolin binding site on the P-glycoprotein is being determined by digestion of labelled protein and immunoprecipitation with peptide specific antibodies. Forskolin binding appears to be associated with the N-terminal and C-terminal halves of the P-glycoprotein. We have also identified a specific region of the N-terminus which is labeled by forskolin photoaffinity labels. In order to study the binding interactions of forskolin and other agents with the P-glycoprotein, studies have been initiated to develop a high affinity ligand binding assay using iodinated derivatives of forskolin. These studies are aimed at 1) providing a better understanding of the drug binding site; 2) developing forskolin derivatives as potential therapeutic agents; and 3) developing forskolin derivatives an potential in vivo diagnostic agents.